Marianne Engeser

Room-Temperature CH Bond Activation of Methane by Bare [P4O10].+†

No need for a metal: A combination of mass spectrometry and computational studies (density functional theory and coupled-cluster methods) shows that [P(4)O(10)](.+) is the first polynuclear nonmetal oxide cation that is capable of activating the C--H bond of methane at room temperature (see picture). This process represents a further example in the reactivity of oxygen-centered radicals.

The Final Steps of Bacillaene Biosynthesis in Bacillus amyloliquefaciens FZB42: Direct Evidence for β,γ Dehydration by a trans-Acyltransferase Polyketide Synthase†

The antibiotic bacillaene (1; Scheme 1) is the prototype of a growing class of polyketides synthesized by a family of polyketide synthases (PKSs) termed trans-acyltransferase (AT) PKSs. 3] These poorly studied enzymes are giant multimodular proteins that have evolved independently from the textbook (cis-AT) PKSs involved in, for example, erythromycin biosynthesis. Since they are rare in actinomycetes, the first model organisms used for studies on polyketide biosynthesis, trans-AT PKSs have long been overlooked. However, they are now known to be widespread among many other rich natural product sources and responsible for the production of several pharmacologically relevant polyketides, including antibiotics of the mupirocin and streptogramin groups and the antitumor drug candidate bryostatin 1. trans-AT PKSs have also attracted considerable ecological interest, since they occur in bacteria with unusual lifestyles, such as symbionts, pathogens, and anaerobes. 6] Another peculiarity that sets them apart from cisAT enzymes and raises fundamental questions about how polyketides are assembled by these proteins is the high frequency of biosynthetic features that do not conform with classical PKS rules. 3,7] During functional studies with 1 as a model, we discovered that deletion of the thioesterase (TE) domain, which releases the assembled polyketide chain from the PKS, results in the production of virtually all intermediates. This unusual phenomenon enabled us to unravel a large part of bacillaene biosynthesis and, in combination with phylogenetic analyses, to establish a set of colinearity rules that can be applied to the prediction and discovery of natural products from trans-AT PKS sequences. Several questions, however, remained unanswered after the studies on 1. The triene system in the C3–C8 region exhibits rare b,g-type unsaturation instead of the standard a,b pattern; the resulting enamide moiety is known in few other polyketides. Since 1 and its advanced precursors are highly unstable, our previous structural investigations to characterize the bacillaene assembly had relied on HPLC coupled to high-resolution MS (HRMS). This technique, however, does not distinguish between constitutional isomers and thus leaves unclear when and how trans-AT PKSs catalyze olefinic shifts (Scheme 1). We also noticed that in late cultures of the bacillaene producer Bacillus amyloliquefaciens FZB42, 1 was almost entirely replaced by a new compound of unknown structure (see Figure S1 in the Supporting Information). This result raised the question of whether 1 is really the true biosynthetic end product. Finally, the bacillaene (bae) PKS terminates with two noncanonical modules (modules 16 and 17, Scheme 1), the ketosynthase (KS) domains of which were predicted by bioinformatic analysis to be unable to catalyze chain-elongation reactions. The role of such seemingly superfluous terminal modules, which are ubiquitous in trans-AT PKSs, is presently unknown. Another architectural oddity is a dehydratase (DH) domain in module 16 which could not be attributed to any dehydration reaction. To examine the function of modules 16 and 17, we genetically engineered the PKS mutants JM54-2, with a deletion of module 17 + TE, and JM122, in which module 17 plus the TE domain are fused directly onto module 15. HPLC–HRMS analysis showed that both strains produced a series of prematurely released intermediates, the two most advanced of which had the molecular formulae C34H48N2O6 [*] J. Moldenhauer, Dr. M. Engeser, Prof. Dr. J. Piel Kekul Institut f r Organische Chemie und Biochemie Universit t Bonn Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) Fax: (+ 49)228-739-712 E-mail: joern.piel@uni-bonn.de Homepage: http:/www.chemie.uni-bonn.de/oc/ak_piel

Light‐Induced Formation of G‐Quadruplex DNA Secondary Structures

The attachment of photolabile “protecting” groups to mask the activity of biologically active compounds is commonly referred to as “caging”. The active molecule can be released by irradiation with the laser of a confocal microscope. The method provides exact control over the location, dose and time at which this event occurs. The strengths of this approach lie, for example, in the arbitrary choice of location where a pro-

Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling.

Cleavage of four carbon-carbon bonds during biosynthesis of the griseorhodin a spiroketal pharmacophore.

The rubromycins, such as gamma-rubromycin, heliquinomycin, and griseorhodin A, are a family of extensively modified aromatic polyketides that inhibit HIV reverse transcriptase and human telomerase. Telomerase inhibition crucially depends on the presence of a spiroketal moiety that is unique among aromatic polyketides. Biosynthetic incorporation of this pharmacophore into the rubromycins results in a dramatic distortion of the overall polyketide structure, but how this process is achieved by the cell has been obscure. To identify the enzymes involved in spiroketal construction, we generated 14 gene-deletion variants of the griseorhodin A biosynthetic gene cluster isolated from the tunicate-associated bacterium Streptomyces sp. JP95. Heterologous expression and metabolic analysis allowed for an assignment of most genes to various stages of griseorhodin tailoring and pharmacophore generation. The isolation of the novel advanced intermediate lenticulone, which exhibits cytotoxic, antibacterial, and elastase-inhibiting activity, provided direct evidence that the spiroketal is formed by cleavage of four carbon-carbon bonds in a pentangular polyketide precursor. This remarkable transformation is followed by an epoxidation catalyzed by an unusual cytochrome P450/NADPH:ubiquinone oxidoreductase pair that utilizes a saturated substrate. In addition, the absolute configuration of griseorhodin A was determined by quantum-chemical circular dichroism (CD) calculations in combination with experimental CD measurements.

Cap for copper(I) ions! Metallosupramolecular solid and solution state structures on the basis of the dynamic tetrahedral [Cu(phenAr2)(py)2]+ motif.

The tetrahedral [Cu(phenAr(2))(py)(2)](+) coordination motif (phen = 1,10-phenanthroline; py = pyridine) conceived on the basis of the HETPYP concept (heteroleptic pyridyl and phenanthroline metal complexes) is a versatile dynamic unit for constructing various heteroleptic metallosupramolecular pseudo-1D, 2D, and 3D structures, both in solution and the solid state. The 2,9-diaryl substituted phenanthroline (phenAr(2)) serves as a capping ligand for copper(I) ions, as its bulky nature prevents formation of the homoleptic complex [Cu(phenAr(2))(2)](+). Combination of the dynamic and concave metal ligand building block [Cu(phenAr(2))](+) with various pyridine (py) ligands, such as bi-, tri-, and tetra-pyridines, opened the way to infinite 1D helicates, 2D networks, and discrete 3D hexanuclear cages, whereas spatial integration of both phenAr(2) and py units into a single ligand resulted in the formation of a Borromean-ring-type hexanuclear cage.

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

Thermal Homo‐ and Heterolytic CH Bond Activation of Ethane and Propane by Bare [P4O10].+: Regioselectivities, Kinetic Isotope Effects, and Density Functional Theory Based Potential‐Energy Surfaces

Mechanistic studies of regioselective bond activation, for example, primary versus secondary C H bonds in hydrocarbons, constitute a classical pursuit in physical organic and computational chemistry. For homolytic C H bond cleavages, brought about by halogen atoms or hydroxyl radicals, varying preferences have been noticed previously in favor of the weaker secondary C H bond. This holds also true in the thermal gas-phase reaction of bare [NiX] (X=F, Cl, Br) with propane [Eq. (1)].

Heteroleptic metallosupramolecular racks, rectangles, and trigonal prisms: stoichiometry-controlled reversible interconversion.

A simple approach toward preparation of heteroleptic two-dimensional (2D) rectangles and three-dimensional (3D) triangular prisms is described utilizing the HETPYP (HETeroleptic PYridyl and Phenanthroline metal complexes) concept. By mixing metal-loaded linear bisphenanthrolines of varying lengths with diverse (multi)pyridine (py) ligands in a proper ratio, six different self-assembled architectures arise cleanly and spontaneously in the absence of any template. They are characterized by (1)H and DOSY NMR, ESI-FT-ICR mass spectrometry as well as by Job plots and UV-vis titrations. Density functional theory (DFT) computations provide information about each structure. A stoichiometry-controlled supramolecule-to-supramolecule interconversion based on the relative amounts of metal bisphenanthroline and bipyridine forces the rectangular assembly to reorganize to a rack architecture and back to the rectangle, as clearly supported by variable temperature and DOSY NMR as well as dynamic light scattering data. The highly dynamic nature of the assemblies represents a promising starting point for constitutional dynamic materials.

A fluorescent molecular thermometer based on the nickel(II) high-spin/low-spin interconversion

The temperature of a fluid can be measured through a variation of the fluorescence emission intensity of a naphthalene fragment covalently linked to a NiII tetraaza-macrocyclic complex, which undergoes a temperature dependent spin interconversion equilibrium.